We have continued our studies of chromatin structure in the neighborhood of expressed genes, making use of the globin gene family in chicken erythrocytes as a model system. We investigated further the enhancer which we had found at the 3, end of the adult beta globin gene. The activity of the enhancer has been analyzed by construction of a series of scanning mutations across the domain, and transfection into primary 9 day embryonic erythrocytes. Of the four previously identified protein-binding regions within the enhancer, only two are important for function in these cells. One of these binds two copies of a novel erythroid - specific factor, which we have named Eryfl. We find that every member of the alpha and beta globin gene families has a site for binding Eryfl in a neighboring regulatory region. An Eryfl site is also present in the 3' enhancer of the human beta globin gene. This protein may play a major role in specifying the genes to be activated during erythroid development. We have also used monoclonal antibodies to identify another erythroid - specific factor, BGP1, which binds to the string of 16 G residues in the beta promoter, and which appears to be related to, but distinct from, the general factor Spl. We have continued the study of the 3' beta enhancer, and shown that it can control the embryonic epsilon promoter, located 5' of the enhancer, with an activity in vitro that partially mimics the stage-specific regulation seen in vivo. We have also begun an analysis of the adult alpha globin genes, and shown that their regulatory mechanisms, which share some elements with the beta family, are nonetheless much simpler. This simplicity is consistent with the limited regulatory repertoire of these genes in vivo.